Regulation of phospholamban and sarcoplasmic reticulum ca<Superscript>2+</Superscript>-atpase by atorvastatin: implication for cardiac hypertrophy

Abnormal intracellular Ca2+ homeostasis in the myocardium has been suggested as the cause of cardiac hypertrophy, and this process can be prevented by the HMG-CoA reductase inhibitors, statins. In the present study, the effect of atorvastatin on left ventricular hypertrophy was investigated, and then whether the underlying mechanism was related to a defect in intracellular Ca2+ homeostasis explored. Twelve spontaneously hypertensive rats (SHR), at 8 weeks old, were used in this study, and received either distilled water or atorvastatin for ten weeks, with age-matched normotensive Wistar-Kyoto rats (WKY) used as controls. RT-PCR and western blotting were used to detect the mRNA and protein expressions of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), respectively, and a colorimetric method used to examine the SERCA2a activity. Additionally, cardiac hypertrophic indices, such as the cardiosomatic ratio, left ventricular weight to body weight (LVW/BW) ratio and cardiomyocytes transverse diameter (TDM), together with the systolic blood pressure (SBP) and serum lipids levels were also examined. After ten weeks, significant decreases were observed in both the mRNA and protein expression levels of SERCA2a, as well as its activity, in the hypertrophied hearts of the SHR. The administration of atorvastatin to the same strains of rats effectively inhibited these decreases, and the above cardiac hypertrophic indices, as well as the SBP and serum lipids levels were significantly decreased. However, no significant changes in the expressions of PLB were observed in WKY, SHR and atorvastatin-treated SHR. These findings demonstrated that through regulation of the PLB and SERCA2a levels in the hearts of SHR, atorvastatin can prevent the cardiac hypertrophy caused due to pressure overload, which provides a relatively new insight into the mechanism of atorvastatin in the prevention of cardiac hypertrophy.     

Role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> content in Ca<Superscript>2+</Superscript> entry of bovine airway smooth muscle cells

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2⁺ channels with methoxyverapamil or by the sustained K⁺-induced depolarisation. The slow Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.     

Regulation of cardiac Ca<Superscript>2+</Superscript> and ion channels by shear mechanotransduction

Cardiac contraction is controlled by a Ca²⁺ signaling sequence that includes L-type Ca²⁺ current-gated opening of Ca²⁺ release channels (ryanodine receptors) in the sarcoplasmic reticulum (SR). Local Ca²⁺ signaling in the atrium differs from that in the ventricle because atrial myocytes lack transverse tubules and have more abundant corbular SR. Myocardium is subjected to a variety of forces with each contraction, such as stretch, shear stress, and afterload, and adapts to those mechanical stresses. These mechanical stimuli increase in heart failure, hypertension, and valvular heart diseases that are clinically implicated in atrial fibrillation and stroke. In the present review, we describe distinct responses of atrial and ventricular myocytes to shear stress and compare them with other mechanical responses in the context of local and global Ca²⁺ signaling and ion channel regulation. Recent evidence suggests that shear mechanotransduction in cardiac myocytes involves activation of gap junction hemichannels, purinergic signaling, and generation of mitochondrial reactive oxygen species. Significant alterations in Ca²⁺ signaling and ionic currents by shear stress may be implicated in the pathogenesis of cardiac arrhythmia and failure.     

Effect of carticaine on the sarcoplasmic reticulum Ca<Superscript>2+</Superscript>-adenosine triphosphatase. II. Cations dependence

Ca²⁺-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca²⁺ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca²⁺] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca²⁺-ATPase activity and the coupled Ca²⁺ uptake by isolated SR vesicles, and increases the rate of Ca²⁺ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca²⁺-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca²⁺ and Mg²⁺. The activating effect of Ca²⁺ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca²⁺ binding to the high affinity Ca²⁺ transport sites. The activating effect of Mg²⁺ on the phosphorylation of Ca²⁺-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca²⁺-ATPase cycle.Drs. Guillermo L. Alonso and Patricia Bonazzola are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República ArgentinaThe experiments were performed in accordance with the current laws of our country     

Inhibitory effect of YM-244769, a novel Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger inhibitor on Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange current in guinea pig cardiac ventricular myocytes

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na⁺/Ca²⁺ exchange (NCX) inhibitor by using various cells transfected with NCX using the ⁴⁵Ca²⁺ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (I_NCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional I_NCX in a concentration-dependent manner. The IC₅₀ values of YM-244769 for the bidirectional outward and inward I_NCX were both about 0.1 μM. YM-244769 suppressed the unidirectional outward I_NCX (Ca²⁺ entry mode) with an IC₅₀ value of 0.05 μM. The effect on the unidirectional inward I_NCX (Ca²⁺ exit mode) was less potent, with 10 μM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na⁺ concentration, YM-244769 suppressed I_NCX more potently than it did at 0 mM [Na⁺]_i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca²⁺ entry mode of NCX more potently than the Ca²⁺ exit mode, and inhibition by YM-244769 is [Na⁺]_i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.     

Effects of trimebutine maleate on colonic motility through Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels and L-type Ca<Superscript>2+</Superscript> channels

The effects of trimebutine maleate (TM) on spontaneous contractions of colonic longitudinal muscle were investigated in guinea pigs. The contractile responses of smooth muscle strips were recorded by an isometric force transducer. Membrane and action potentials were detected by an intracellular microelectrode technique. The whole-cell patch clamp recording technique was used to record the changes in large conductance Ca²⁺-activated K⁺ (BK_ca) and L-type Ca²⁺ currents in colonic smooth muscle cells. At high concentrations (30, 100, and 300 μM), TM inhibited the amplitude of spontaneous contractions. At low concentrations (1 and 10 μM), TM attenuated the frequency and tone of smooth muscle strips, whereas TM had no influence on the amplitude of spontaneous contractions. TM depolarized the membrane potentials, but decreased the amplitude and frequency of action potentials at high concentrations. TM inhibited BK_ca and L-type Ca²⁺ currents in a dose-dependent manner. In the presence of the BK_ca channel opener, NS1619, TM also inhibited BK_ca currents. Bayk8644, a L-type Ca²⁺ channel opener, increased L-type Ca²⁺ currents. This augmentation was also attenuated by TM. These results suggest that TM attenuates intestinal motility through inhibition of L-type Ca²⁺ currents, and depolarizes membrane potentials by reducing BK_ca currents. Thus, TM may be a multiple-ion channel regulator in the gastrointestinal tract.     

Magnesium isoglycyrrhizinate inhibits L-type Ca<Superscript>2+</Superscript> channels,Ca<Superscript>2+</Superscript> transients,and contractility but not hERG K<Superscript>+</Superscript> channels

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K⁺ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca²⁺ currents (I_Ca-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (I_Kr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited I_Ca-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 0.22 mg/ml, and the maximal inhibitory effect was 61.10 ± 0.59%. MI at a concentration of 0.3 mg/ml reduced cell shortening by 24.12 ± 3.97% and the peak value of the Ca²⁺ transient by 36.54 ± 4.96%. MI had no significant influence on hERG K⁺ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of I_Ca-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on I_Kr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.     

Store operated Ca<Superscript>2+</Superscript> influx by selective depletion of ryanodine sensitive Ca<Superscript>2+</Superscript> pools in primary human skeletal muscle cells

The contraction and relaxation of skeletal muscle is driven by release of Ca2+ from sarcoplasmic reticulum through the ryanodine receptor type 1 and extruding the ion from the cytosol by Ca2+ ATPases. Efficient refilling of the empty Ca2+ stores is essential for repetitive cycles of muscle contraction and relaxation, but not investigated in human skeletal muscle cells. Here we show that under conditions of selective depletion of the ryanodine-sensitive Ca2+ pool Ca2+ influx occurs in differentiated human skeletal muscle cells using the Ca2+ imaging technique. This Ca2+ influx is not due to permeation through the L-type Ca2+ channel and not observed under conditions of inhibited Ca2+ ATPase. The Ca2+ influx was visualised by quenching the intracellular fura2 signal with Mn2+ on single cell level and also using fluorescence photometry of cell suspensions. The Mn2+ influx was inhibited by the Ca2+ channel blockers La(3+) and SKF96356. The delineation of the signalling cascade leading to Ca2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores showed that phospholipase C or protein kinase C were not involved. Interestingly, a Mn2+ influx was triggered by the cell-permeant analogue of diacylglycerol and further augmented by the application of RHC80267, a diacylglycerol lipase inhibitor. This signalling pathway could be attributed to the participation of a protein kinase C activity. However, Mn2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores was not altered by RHC80267 or protein kinase C inhibitors. Using RT-PCR, correctly spliced mRNA fragments were detected corresponding to human transient receptor potential (TRPC) Ca2+ channels type 1, 3, 4 and 6. These data show that in skeletal muscle at least two independent mechanisms of Ca2+ influx exist. For Ca2+ influx triggered by the selective depletion of ryanodine sensitive Ca2+ stores we propose a phospholipase C independent coupling of ryanodine receptors to voltage insensitive Ca2+ channels.     

Effect of nitroxyalkyl derivatives of fullerenylproline on the activity of CA<Superscript>3+</Superscript>-ATPase of sarcoplasmic reticulum

The effect of nitroxyalkyl derivatives of fullerenylproline methyl ester on the enzymatic activity of Ca²⁺-ATPase of sarcoplasmic reticulum (SR) has been studied. It is shown that hybrid derivatives of C₆₀ fullerene are capable of inhibiting the activity of Ca²⁺-ATPase of SR. The mononitrate inhibits the hydrolytic activity of the enzyme with K _i = 1.92 × 10⁻⁶ M; active Ca²⁺ transport, with K _i = 3.79 × 10⁻⁶ M. The dinitrate inhibits ATP hydrolysis with K _i = 2.38 × 10⁻⁸ M; Ca²⁺ transport, with K _i  = 3.08 × 10⁻⁸ M. Fullerenylproline methyl ester does not affect the enzymatic activity of Ca²⁺-ATPase. Based on these data it is possible to predict the possible fields of application for hybrid fullerene derivatives as potential drugs.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

Analysis of vasopressin-induced Ca<Superscript>2+</Superscript> increase in rat hepatocytes

To analyze vasopressin-induced Ca2+ increase in liver cells, rat hepatocytes were isolated and attached to collagen-coated cover slips. Using fura-2, a Ca2+-sensing dye, changes in intracellular Ca2+ concentration by vasopressin were monitored. Results in this communication suggested that vasopressin-induced Ca2+ increase were composed of both Ca2+ release from internal Ca2+ stores and influx from the plasma membrane. The Ca2+ influx consisted of two distinguishable components. One was dependent on the presence of vasopressin and the other was not. SK&F96365 blocked vasopressin-induced Ca2+ influx in a dose-dependent manner. Vasopressin-induced Ca2+ release from internal stores diminished in a primary culture of hepatocytes according to the culture time. However, changes in vasopressin-induced Ca2+ influx across the plasma membrane differed from changes in the Ca2+ release from internal stores, suggesting two separate signalings from receptor activation to internal stores and to the plasma membrane.     

Reconstitution of the cytoplasmic interaction between phospholamban and Ca(2+)-ATPase of cardiac sarcoplasmic reticulum

Phospholamban (PLN) reversibly inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca(2+) in heart muscle cells. The interaction between PLN and SERCA2a occurs at multiple sites within the cytoplasmic and membrane domains. Here, we have reconstituted the cytoplasmic protein-protein interaction using bacterially expressed fusion proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of SERCA2a. We have developed two methods to evaluate the binding of the fusion proteins, one with glutathione-Sepharose beads and the other with a 96-well plate. Essentially the same results were obtained by the two methods. The affinity of the binding (K(D)) was 0.70 microM. The association was inhibited by cAMP-dependent phosphorylation of the PLN fusion protein and by usage of anti-PLN monoclonal antibody. It was also diminished by substitution at the phosphorylation site of PLN of Ser(16) to Asp. These results suggest that PLN can bind SERCA2a in the absence of the membrane domains and that the modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel the disruption of the association between the two fusion proteins. It has been shown that the removal of PLN inhibition of SERCA2a rescues cardiac function and morphology in the mouse dilated cardiomyopathy model. Our assay system can be applied to the screening of novel inotropic agents that remove the inhibition of SERCA2a by PLN, improving the relaxation as well as the contractility of the failing heart.     

Roles of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger isoforms NCX1 and NCX2 in motility in mouse ileum

The Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter that is involved in regulating intracellular Ca²⁺ concentrations in various tissues. The physiological roles by which NCX influences gastrointestinal motility are incompletely understood, although its role in the heart, brain, and kidney has been widely investigated. In this study, we focused on the functions of the NCX isoforms, NCX1 and NCX2, in the motility of the ileum in the gastrointestinal tract. We investigated the response to electric field stimulation (EFS) in the longitudinal smooth muscle of the ileum obtained from wild-type mice (WT), NCX1-heterozygote knockout mice (NCX1 HET), NCX2 HET and smooth muscle-specific NCX1.3 transgenic mice (NCX1.3 Tg). EFS induced a phasic contraction that persisted during EFS and a tonic contraction that occurred after the end of EFS. We found that the amplitudes of the phasic and tonic contractions were significantly smaller in NCX2 HET, but not in NCX1 HET, compared to WT. Moreover, the magnitudes of acetylcholine (ACh)- and substance P (SP)-induced contractions of NCX2 HET, but not of NCX1 HET, were smaller compared to WT. In contrast, the amplitudes of the phasic and tonic contractions were greater in NCX1.3 Tg compared to WT. Similar to EFS, the magnitude of ACh-induced contraction was greater in NCX1.3 Tg than in WT. Taken together, our findings indicated that NCX1 and NCX2 play important roles in ileal motility and suggest that NCX1 and NCX2 regulate the motility in the ileum by controlling the sensitivity of smooth muscles to ACh and SP.     

缬沙坦对扩张型心肌病心衰大鼠肌浆网Ca2+-ATP酶及其调控蛋白的影响

目的探讨血管紧张素Ⅱ1型受体阻滞剂（ARB）缬沙坦对扩张型心肌病（DCM）心衰大鼠心肌肌浆网Ca^2＋-ATP酶（SER-CA2a）及其调控蛋白（PLB）的影响及意义。方法腹腔注射多柔比星建立SD大鼠DCM模型。随机分为DCM组（M组,n=11）、缬沙坦组（V组,n=10）、另设正常对照组（C组,n=10）,V组予以缬沙坦30mg·k^-1·d^-1,C组和M组予以生理盐水灌胃,8周后行血流动力学检测,采用RT—PCR和Western—blot法分别检测心肌组织SERCA2a、PLB的mRNA和蛋白含量。结果与正常对照组相比,DCM组左心室压峰值（LVSP）、左室最大压力上升及下降速度（±dp／dtmax）显著下降（均P〈0．05）,左室舒张末压（LVEDP）显著上升（P〈0．05）;缬沙坦组LVSP、±dp／dtmax显著高于DCM组（均P〈0．05）,LVEDP显著低于DCM组（P〈0．05）;DCM组SERCA2a的mRNA及蛋白含量显著低于正常对照组（均P〈0．05）,PLB的mRNA及蛋白含量显著高于正常对照组（均P〈0．05）。缬沙坦治疗后SERCA2a的mRNA及蛋白含量显著升高（均P〈0．05）,PLB的mRNA含量显著降低（P〈0．05）,而PLB蛋白含量无显著改变（P〉0．05）。结论缬沙坦改善DCM心功能可以与其部分纠正SERCA2a、PLB的异常有关。     

L-type Ca<Superscript>2+</Superscript> channels activation and contraction elicited by myricetin on vascular smooth muscles

The effects of myricetin (3,3,4,5,5,7-hesahydroxyflavone), a natural flavonoid found in edible plants, were studied on vascular smooth muscle L-type Ca²⁺ channels by comparing its mechanical, radioligand binding, and electrophysiological properties to those of the Ca²⁺ channel agonist (S)-(-)-Bay K 8644.In rat aorta rings, both myricetin and (S)-(-)-Bay K 8644 induced contractile responses, which were dependent upon prior exposure to K⁺. At 15 mM K⁺ (K15) the pEC₅₀ values for myricetin and (S)-(-)-Bay K 8644 were 4.43±0.03 and 7.92±0.13, respectively. Furthermore, the maximum tension response to myricetin was not significantly different from that elicited by either (S)-(-)-Bay K 8644 or K60. The Ca²⁺ channel blockers nifedipine, verapamil and diltiazem antagonised and fully reverted myricetin-, (S)-(-)-Bay K 8644- as well as K60-induced contractions. Both myricetin and (S)-(-)-Bay K 8644 potentiated rat aorta ring responses to K⁺, shifting the K⁺ concentration-response curve to the left. (S)-(-)-Bay K 8644, but not myricetin, inhibited in a concentration-dependent manner (+)-[³H]PN200–110 binding in porcine aortic membranes. Electrophysiological recordings from single rat tail artery myocytes, under amphotericin B-perforated as well as conventional methods, showed that both myricetin and (S)-(-)-Bay K 8644 increased L-type Ba²⁺ current (I_Ba(L)) and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarising direction, without, however, modifying the threshold potential. Furthermore, (S)-(-)-Bay K 8644 accelerated both activation and inactivation kinetics of I_Ba(L) while myricetin slowed down the activation kinetics. Finally, both (S)-(-)-Bay K 8644 and myricetin slowed down deactivation kinetics of I_Ba(L).These results suggest that myricetin induces vasoconstriction by activating L-type Ca²⁺ channel with similar efficacy but a site of action different to that of (S)-(-)-Bay K 8644.Abbreviations I_Ba(L) L-type Ba²⁺ current - PSS Physiological salt solution - V_h Holding potential     

Regulation of RYR2 by sarcoplasmic reticulum Ca2+

Ca²⁺ is arguably the most important ion involved in the contraction of the heart. The cardiac ryanodine receptor (RyR2), the major Ca²⁺ release channel located in the sarcoplasmic reticulum (SR) membrane, is responsible for releasing the bulk of Ca²⁺ required for contraction. Moreover, RyR2 is also crucial for maintaining SR Ca²⁺ homeostasis by releasing Ca²⁺ from the SR when it becomes overloaded with Ca²⁺. During normal contraction, RyR2 is activated by cytosolic Ca²⁺, whereas during store overload conditions, the opening of RyR2 is governed by SR Ca²⁺. Although the process of the cytosolic control of RyR2 is well established, the molecular mechanism by which SR luminal Ca²⁺ regulates RyR2 has only recently been elucidated and remains controversial. In addition to the activation of RyR2, SR luminal Ca²⁺ also determines when the RyR2 channel closes. RyR2‐mediated Ca²⁺ release from the SR does not continue until the SR is completely depleted. Rather, it ceases when SR luminal Ca²⁺ falls below a certain level. Given the importance of SR Ca²⁺, it is not surprising that the SR luminal Ca²⁺ level is tightly controlled by SR Ca²⁺‐buffering proteins. Consequently, the opening and closing of RyR2 is heavily influenced by the presence of such proteins, particularly those associated with RyR2, such as calsequestrin and the histidine‐rich Ca²⁺‐binding protein. These proteins appear to indirectly alter RyR2 activity by modifying the microdomain SR Ca²⁺ level surrounding RyR2.     

The role of major apoptotic proteins in Ca<Superscript>2+</Superscript> dependent cell homeostasis

The key role of Bcl-2 (B-cell lymphoma) family proteins in programmed cell death (apoptosis) is reliably established, but particular molecular mechanisms of downregulation involving these anti-and pro-apoptotic proteins are still insufficiently clear. There are several hypotheses explaining the cell-protective (Bcl-2, Bcl-X_L) and pro-apoptotic (Bcl-x, Bak) functions of Bcl-2 family proteins. This paper briefly summarizes the available information concerning the molecular mechanisms of action of the pro-and anti-apoptotic proteins of Bcl-2 family. In particular, an original hypothesis is put forward according to which homo-and heterodimerization of these proteins are the key events in the mechanism of Ca²⁺ ion dependent homeostasis regulation. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 12, pp. 3–8, December, 2005.     

Release of renal dipeptidase from Glycosylphosphatidylinositol anchor by insulin-triggered phospholipase c/intracellular Ca<Superscript>2+</Superscript>

Glycosylphosphatidylinositol (GPI) anchored proteins appear to be released from the plasma membrane due to various extracellular stimuli. To determine the signaling pathway from insulin to GPI-protein, the release of GPI-renal dipeptidase (RDPase, EC 3.4.13.19) from porcine proximal tubules, stimulated by insulin, was explored. Insulin stimulated the release of RDPase in a concentration-dependent manner (half maximal release at 0.58 nM), which peaked at 10-20 min. Western blot analysis, with antibody against the cross-reacting determinant (CRD), revealed that RDPase was released by a GPI-specific phospholipase C (GPI-PLC), and was shown to be Ca2+-dependent. A PI-PLC inhibitor, U73122, effectively blocked the effect of insulin on the release of RDPase, suggesting insulin is associated with an intracellular PI-PLC. Insulin treatment increased the production of intracellular Ca2+ from porcine proximal tubules. Intracellular Ca2+, coupled with insulin, facilitated the releases of RDPase, an inhibitor of inositol trisphosphate-dependent Ca2+ from the endoplasmic reticulum, and a Ca2+ channel blocker that blocked the effect of insulin. Taken together, these results suggest that insulin, in part, may activate a GPI-PLC, via PI-PLC/intracellular Ca2+, which may consequently stimulate the release of RDPase.     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 20 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.